Genetically-engineered mouse models of small cell lung cancer: the next generation

Abstract

Small cell lung cancer (SCLC) remains the most fatal form of lung cancer, with patients in dire need of new and effective therapeutic approaches. Modeling SCLC in an immunocompetent host is essential for understanding SCLC pathogenesis and ultimately discovering and testing new experimental therapeutic strategies. Human SCLC is characterized by near universal genetic loss of the RB1 and TP53 tumor suppressor genes. Twenty years ago, the first genetically-engineered mouse model (GEMM) of SCLC was generated using conditional deletion of both Rb1 and Trp53 in the lungs of adult mice. Since then, several other GEMMs of SCLC have been developed coupling genomic alterations found in human SCLC with Rb1 and Trp53 deletion. Here we summarize how GEMMs of SCLC have contributed significantly to our understanding of the disease in the past two decades. We also review recent advances in modeling SCLC in mice that allow investigators to bypass limitations of the previous generation of GEMMs while studying new genes of interest in SCLC. In particular, CRISPR/Cas9-mediated somatic gene editing can accelerate how new genes of interest are functionally interrogated in SCLC tumorigenesis. Notably, the development of allograft models and precancerous precursor models from SCLC GEMMs provides complementary approaches to GEMMs to study tumor cell-immune microenvironment interactions and test new therapeutic strategies to enhance response to immunotherapy. Ultimately, the new generation of SCLC models can accelerate research and help develop new therapeutic strategies for SCLC.

Fig. 1. Frequently used SCLC GEMMs developed using classical mouse genetics.

A The RP model is generated by intratracheally injecting adeno-Cre into the lungs of mice that harbor homozygous Rb1^flox/flox and Trp53^flox/flox alleles. Intranasal delivery is an alternative route for delivery, but can induce olfactory tumors in some contexts, so intratracheal injection is preferred. B The RPR2 model is generated by intratracheally injecting adeno-Cre into the lungs of mice that harbor homozygous Rb1^flox/flox, Trp53^flox/flox, Rbl2^flox/flox alleles. The “classic” SCLC histology of the RPR2 GEMM is shown on the right (hematoxylin and eosin counterstain). C The RPM model is generated by intratracheally injecting adeno-Cre into the lungs of mice that harbor homozygous Rb1^flox/flox, Trp53^flox/flox alleles, and transgenic loxP-Stop-loxP Myc^T58A-Ires-Luciferase knocked into the H11b locus. The creators and subsequent investigators have noted that while the Myc allele harbors the gene coding for luciferase, this allele has not proven reliable for quantitative bioluminescent imaging; however, the Luc transcript can be utilized in single-cell transcriptomics and potentially for other applications. The “variant” SCLC histology of the RPM GEMM is shown on the right. However, the RPM GEMM has histological variation from “classic” in situ lesions toward more invasive tumors. Scale bar, 100 μm.

Fig. 2. Approaches to make the “Next Generation” of SCLC GEMMs using CRISPR/Cas9 somatic engineering.

A The all-in-one adenoviral approach to make CRISPR/Cas9 SCLC GEMMs using LSL-Cas9 mice where the adenovirus contains sgRNAs targeting Rb1, Trp53, Rbl2, and a gene of interest (sgT) as well as CMV-Cre. B Examples of insertion or deletions (indels) in Rb1, Trp53, Rbl2, and sgT (Kdm5a) from mouse SCLC lung tumors. C The CRISPR/Cas9/Cre hybrid adenoviral approach to make CRISPR/Cas9 SCLC GEMMs using RPR2 mice where the adenovirus contains sgT, Cas9, and Cre. D The CRISPR/Cas9 adenoviral or lentiviral approach to make CRISPR/Cas9 SCLC GEMMs using RP, RPR2, or RPM LSL-Cas9 mice where the adenovirus contains sgT and Cre. A, C, and D were created with BioRender.com.

Fig. 3. Approaches to generate immunocompetent syngeneic mouse models of SCLC.

A Schematic showing the process of developing syngeneic immunocompetent mouse models of SCLC (allografts) from autochthonous SCLC GEMMs. B Possible injection sites to grow syngeneic SCLC models are shown including intracranial, intrathoracic, intratracheal, subcutaneous, and tail vein. C Schematic showing the process of developing precancerous SCLC mouse models (preSCs) using SCLC GEMMs with premalignant lesions. Note that while C57BL/6 mice are often used for immunocompetent models, some of the historical Rb1 and Trp53 alleles were bred into the 129Sv/J background; cancer cell lines derived from these models can often be grown in F1 mice from C57BL/6 and 129Sv/J parents. A, B, and C were created with BioRender.com.